Synthesis and use of isotopically labeled macrocyclic compounds

ABSTRACT

Disclosed herein are isotopically labeled antifungal antibiotics and related compounds. Also disclosed are methods for synthesizing these isotopically labeled molecules and using the same to study the distribution of these compounds in the biosphere as well as the products formed by the breakdown of these isotopically labeled compounds.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication Ser. No. 61/747,699 filed Dec. 31, 2012, which is expresslyincorporated by reference herein.

FIELD OF THE INVENTION

Aspects of the invention include the preparation and use of isotopicallylabeled compounds including radiolabeled forms of the antifungalantibiotic UK-2A and related compounds.

BACKGROUND OF THE INVENTION

Isotopically labeled compounds are important tools in characterizing therole and fate of bioactive molecules in living systems and inecosystems. Determining the mechanism of action and the fate ofbioactive molecules may be instrumental in winning its approval for useas a crop protection chemical and in developing new generations ofmolecules that may have properties that are superior to those of thestarting molecule. This process often times involves following the fateof a novel molecule or existing molecules being evaluated for new orexpanded utility in the biosphere. For example, plants can be treatedwith appropriately labeled forms of the compounds under study. Bytracking the fate of radiolabeled portions of the molecule it ispossible to track the distribution of the compounds and moieties formedby the breakdown of these labeled compounds.

Such studies can be used to determine if particular molecules are phloemmobile, that is if they or possible dangerous compounds formed from themetabolism of these molecules can find their way into edible portions ofthe plants. Still other information that can be gleaned using thisapproach, including tracking the uptake of these molecules into variousplants, fungi or insects in the biosphere.

Appropriately isotopically labeled versions of agriculturally activecompounds can also be used to help identify the metabolites of thesecompounds. Of course, before these radiolabeled molecules can be usedthey must first be synthesized. The chemical or in some instancesbio-synthesis of properly labeled bioactive molecules is sometimesextremely challenging. These syntheses include both the normalchallenges of working with complex molecules and in some caseschemically toxic compounds, and the additional complication of usingradioactive starting materials. These starting materials, in addition togenerating radioactive products, by products, equipment, solvents, andthe like, often require unique synthetic approaches to incorporate theisotope into the agriculturally active compound. Some of the materialsand methods disclosed herein include methods of making and using suchradiolabeled compounds.

SUMMARY OF THE INVENTION

Some aspects of the present invention are methods for the preparationand use of radiolabeled macrocyclic compounds including radiolabeledanalogues and components of the antifungal antibiotic UK-2A.

Some aspects of the invention include radiolabeled compounds of thefollowing formula:

wherein R₁ is selected from the group consisting of

-   -   H and

R₂ is selected from the group consisting of

-   -   OH and

and R₄ is selected from the group consisting of phenyl (Ph), (¹³C₆)Ph,and (UL-¹⁴C)Ph, wherein UL is defined as uniformly labeled.

In some embodiments of the invention R₁ is

In still other embodiments R₂ is

And in some embodiments R₁ is H.

Some aspects of the invention include isotopically labeled compoundsaccording to the following formula:

wherein R₃ is selected from the group consisting of

and R₄ is selected from the group consisting of (¹³C₆)Ph and (UL-¹⁴C)Ph.

In some embodiments of the invention R₃ is

In still other embodiments R₃ is

In yet other embodiments R₃ is

And in some embodiments R₄ is (¹³C₆)Ph or (UL-¹⁴C)Ph.

Some aspects of the invention include methods for studying the biospherecomprising the steps: analyzing samples of materials exposed to at leastone isotopically labeled compound according to the instant disclosureand determining which samples include an isotopic signature from saidcompound. In some embodiments the sample is a portion of a plant. Insome embodiments the sample is from a material that is a surfaceadjacent to a plant or connected to a surface adjacent to a plant. Insome embodiments of the invention the isotopic signature is associatedwith a metabolite or degradation product of said compound.

In one exemplary embodiment, a radiolabeled compound is of the formula:

wherein R₁ is selected from the group consisting of: H and

R₂ is selected from the group consisting of: OH and

and R₄ is selected from the group consisting of Ph, Ph-¹³C₆, andPh-UL-¹⁴C. In a more particular embodiment, R₁ is

In another more particular embodiment, R₁ is H. In still another moreparticular embodiment, R₂ is:

In one exemplary embodiment, an isotopically labled compound is of theformula:

wherein R₃ is selected from the group consisting of: H,

and R₄ is at least one atom selected from the group consisting ofPh-¹³C₆, and Ph-UL-¹⁴C. In a more particular embodiment, R₃ is

In another more particular embodiment, R₃ is:

In still another more particular embodiment, R₃ is:

In still yet another more particular embodiment, R₄ is Ph-¹³C₆. Inanother more particular embodiment, R₄ is Ph-UL-¹⁴C.

In one exemplary embodiment, a method for studying the biosphere isprovided. The method includes analyzing one or more samples exposed toan isotopically labeled compound of the formula:

wherein R₁ is selected from the group consisting of H and

R₂ is selected from the group consisting of OH and

and R₄ is selected from the group consisting of Ph, Ph-¹³C₆, andPh-UL-¹⁴C. The method further includes determining which samples, ifany, include an isotopically labeled signature from said compound. In amore particular embodiment, the sample is a portion of a plant. Inanother more particular embodiment, the sample is from a surfaceadjacent to a plant. In still another more particular embodiment, thesample is from a material that is in communication with a surfaceadjacent to a plant. In yet still another more particular embodiment,the isotopically labeled signature is contained in a portion of saidcompound.

In one exemplary embodiment, another method for studying the biosphereis provided. The method includes analyzing one or more samples exposedto an isotopically labeled compound of the following formula:

wherein R₃ is selected from the group consisting of:

and R₄ is at least one atom selected from the group consisting ofPh-¹³C₆, and Ph-UL-¹⁴C. The method further includes determining whichsamples, if any, include an isotopically labeled signature from saidcompound. In a more particular embodiment, the sample is a portion of aplant. In another more particular embodiment, the sample is from asurface adjacent to a plant. In still another more particularembodiment, the sample is from a material that is in communication witha surface adjacent to a plant. In yet still another more particularembodiment, the isotopically labeled signature is contained in a portionof said compound.

In one exemplary embodiment, a process for the preparation ofradiolabeled compounds of the Formula:

is provided, wherein R₁ is selected from the group consisting of H and

and R₂ is

The process includes (a) contacting a solution of a compound of FormulaA in an aprotic solvent with a compound of Formula B, where Formula Ais:

and Formula B is:

followed by the sequential treatment with an organic amine base and apeptide coupling reagent to produce a compound of Formula C:

The process further includes (b) contacting a solution of thepicolinamide of Formula C in a polar solvent with an alkali carbonatebase, followed by treatment with iodomethyl isobutyrate to produce acompound of Formula D:

In a more particular embodiment, the aprotic solvent is CH₂Cl₂, theorganic amine base is a mixture of 4-methylmorpholine and DMAP, and thepeptide coupling reagent is HATU in step (a). In another more particularembodiment, the polar solvent is acetone and the alkali carbonate baseis Na₂CO₃ in step (b).

In one exemplary embodiment, a process for the preparation ofisotopically labeled compounds of the formula:

is provided, wherein R₃ is selected from the group consisting of: H,

The process includes (c) contacting a preformed mixture of a finelyground metal treated with iodine in a polar, aprotic solvent with asolution of the compound of Formula E:

in a polar, aprotic solvent, followed by treatment with(¹³C₆)-iodobenzene and a palladium catalyst at an elevated temperatureto produce a compound of Formula F:

The process further includes (d) contacting a solution of theBoc-protected aminolactone of Formula F in a polar, aprotic solvent witha solution of a mineral acid in a polar, aprotic solvent to produce acompound of Formula G:

The process further includes (e) contacting a solution of the aminehydrochloride of Formula G in an aprotic solvent with an inorganic,alkali carbonate base or an organic amine base to produce a compound ofFormula H

The process further includes (f) contacting a solution of the aminomacrocycle of Formula H in an aprotic solvent, generated as described instep (e) or in situ, with a compound of Formula I:

followed by treatment with an organic amine base and a peptide couplingreagent to produce a compound of Formula J:

The process further includes contacting a solution of the picolinamideof Formula J in a polar solvent with an alkali carbonate base followedby iodomethyl isobutyrate to produce a compound of Formula K:

In a more particular embodiment, in step (c) the finely ground metal iszinc dust, the polar aprotic solvent is DMF, the palladium catalyst is(PdCl₂[P(o-Tol)₃]₂), and the temperature is about (i) 40-50° C. for 30minutes followed by about (ii) 50-60° C. for 60 minutes. In another moreparticular embodiment, in step (d), the mineral acid is hydrogenchloride and the polar, aprotic solvent is dioxane. In another moreparticular embodiment, in step (e), the aprotic solvent is CH₂Cl₂, thealkali carbonate base is NaHCO3 and the organic amine base is4-methylmorpholine. In another more particular embodiment, in step (f),the organic amine base is a mixture of 4-methylmorpholine and catalyticDMAP, the peptide coupling reagent is HATU, and the aprotic solvent isCH₂Cl₂. In another more particular embodiment, in step (g), the alkalicarbonate base is K₂CO₃ and the polar solvent is acetone.

In one exemplary embodiment, a process of the preparation ofradiolabeled compounds of the Formula:

is provided, wherein R₃ is selected from the group consisting of: H,

The process includes (h) contacting a preformed mixture of a finelyground metal treated with iodine in a polar, aprotic solvent with asolution of the compound of Formula E:

in a polar, aprotic solvent, followed by treatment with(UL-¹⁴C)-iodobenzene and a palladium catalyst at an elevated temperatureto produce a compound of Formula L:

The process further includes (i) contacting a solution of theBoc-protected aminolactone of Formula L in a polar, aprotic solvent witha solution of a mineral acid in a polar, aprotic solvent to produce acompound of Formula M:

The process further includes (j) contacting a solution of the aminehydrochloride of Formula M in an aprotic solvent with an inorganic,alkali carbonate base or an organic amine base to produce a compound ofFormula N:

The process further includes (k) contacting a solution of the aminomacrocycle of Formula N in an aprotic solvent, generated as described instep (e) or in situ, with a compound of Formula I:

followed by a peptide coupling reagent and an organic amine base toproduce a compound of Formula O:

The process further includes (1) contacting a solution of thepicolinamide of Formula O in a polar solvent with an alkali carbonatebase followed by iodomethyl isobutyrate to produce a compound of FormulaP:

In a more particular embodiment, in step (h) the finely ground metal iszinc dust, the polar aprotic solvent is DMF, the palladium catalyst is(PdCl₂[P(o-Tol)₃]₂), and the temperature is about (i) 40-50° C. for 30minutes followed by about (ii) 50-60° C. for 120 minutes. In anothermore particular embodiment, in step (i), the mineral acid is hydrogenchloride and the polar, aprotic solvent is dioxane. In another moreparticular embodiment, in step (j), the aprotic solvent is CH₂Cl₂, thealkali carbonate base is NaHCO₃ and the organic amine base is4-methylmorpholine. In another more particular embodiment, in step (k),the organic amine base is a mixture of 4-methylmorpholine and catalyticDMAP, the peptide coupling reagent is HATU, and the aprotic solvent isCH₂Cl₂. In another more particular embodiment, in step (l), the alkalicarbonate base is K₂CO₃ and the polar solvent is acetone.

DETAILED DESCRIPTION OF THE INVENTION

For the purposes of promoting an understanding of the principles of thenovel technology, reference will now be made to the preferredembodiments thereof, and specific language will be used to describe thesame. It will nevertheless be understood that no limitation of the scopeof the novel technology is thereby intended, such alterations,modifications, and further applications of the principles of the noveltechnology being contemplated as would normally occur to one skilled inthe art to which the novel technology relates are within the scope ofthis disclosure and the claims.

As used herein, unless explicitly stated otherwise or clearly impliedotherwise the term ‘about’ refers to a range of values plus or minus 10percent, e.g. about 1.0 encompasses values from 0.9 to 1.1.

The present invention concerns the preparation of radiolabeled3-hydroxy-4-methoxypicolinic acid. (2-¹⁴C)pyridin-3-ol (Compound 2) wasprepared by the method described in J Labelled Comp Rad 1992, 31, 615.The radiolabeled pyridin-3-ol was then converted to the novel3-hydroxy-4-methoxy(2-¹⁴C)pyridine-2-carboxylic acid (Compound 6) usingthe method described in Tetrahedron Lett. 1998, 39, 4363 andsubstantially modified by using radiolabeled starting material.

Some embodiments include the synthesis of(3S,6S,7R,8R)-8-benzyl-3-({[3-hydroxy-4-methoxy(2-¹⁴C)pyridin-2-yl]carbonyl}amino)-6-methyl-4,9-dioxo-1,5-dioxonan-7-yl-2-methylpropanoate(Compound 8) by contacting3-hydroxy-4-methoxy(2-¹⁴C)pyridine-2-carboxylic acid (Compound 6) with(3S,6S,7R,8R)-8-benzyl-3-[(tert-butoxycarbonyl)amino]-6-methyl-4,9-dioxo-1,5-dioxonan-7-yl2-methylpropanoate (Compound 7).

The present invention concerns the preparation of(3S,6S,7R,8R)-8-benzyl-3-({[4-methoxy-3-{[(2-methylpropanoyl)oxy]methoxy}(2-¹⁴C)pyridin-2-yl]carbonyl}amino)-6-methyl-4,9-dioxo-1,5-dioxonan-7-yl2-methylpropanoate(Compound 9) by contacting(3S,6S,7R,8R)-8-benzyl-3-({[3-hydroxy-4-methoxy(2-¹⁴C)pyridin-2-yl]carbonyl}amino)-6-methyl-4,9-dioxo-1,5-dioxonan-7-yl-2-methylpropanoate(Compound 8) with iodomethyl isobutyrate.

Another embodiment of the invention concerns the conversion of UK-2A toCompound 7 by treating with di-tert-butyl dicarbonate (BOC anhydride),4-dimethylamino pyridine (DMAP) and N,N-diethylethylenediamine.

In another step of the present invention(3S,6S,7R,8R)-8-benzyl-3-((tert-butoxycarbonyl)amino)-6-methyl-4,9-dioxo-1,5-dioxonan-7-ylisobutyrate (Compound 7) undergoes selective oxidation of the phenylring using sodium periodate (NaIO₄) and a catalytic amount of ruthenium(III) chloride (RuCl₃) to give2-((3S,7R,8R,9S)-3-((tert-butoxycarbonyl)amino)-8-(isobutyryloxy)-9-methyl-2,6-dioxo-1,5-dioxonan-7-yl)aceticacid (Compound 10).

In yet another step of the present invention,2-((3S,7R,8R,9S)-3-((tert-butoxycarbonyl)amino)-8-(isobutyryloxy)-9-methyl-2,6-dioxo-1,5-dioxonan-7-yl)aceticacid (Compound 10) was treated with isobutyl chloroformate followed by2-mercaptopyridine-N-oxide to form the intermediate Barton ester.Hunsdiecker iodination was then accomplished by irradiating the Bartonester with a 450 watt (W) mercury (Hg) lamp in the presence of iodoform(CHI₃) in a quartz reactor to provide the key iododmethyleneintermediate,(3S,6S,7R,8R)-3-((tert-butoxycarbonyl)amino)-8-(iodomethyl)-6-methyl-4,9-dioxo-1,5-dioxonan-7-ylisobutyrate (Compound 11).

The following examples are presented to illustrate the invention, theseexamples are presented by way of illustration and not limitation.

SCHEMES AND EXAMPLES

Example 1, Steps 1 Through 6 Preparation of3-hydroxy-4-methoxy(2-¹⁴C)pyridine-2-carboxylic acid (Compound 6)

(2-¹⁴C)pyridin-3-ol (Compound 2) was prepared by the method described inJ Labelled Comp Rad 1992, 31, 615. The radiolabeled pyridin-3-ol wasthen converted to 3-hydroxy-4-methoxy(2-¹⁴C)pyridine-2-carboxylic acid(Compound 6) using the method described in Tetrahedron Lett. 1998, 39,4363.

Example 1, Steps 7a Preparation of(3S,6S,7R,8R)-8-benzyl-3-[(tert-butoxycarbonyl)amino]-6-methyl-4,9-dioxo-1,5-dioxonan-7-yl2-methylpropanoate (Compound 7)

To a dry 2000 milliliter (mL) round bottom flask equipped withmechanical stirrer, nitrogen (N₂) inlet, addition funnel, andthermometer, was charged(3S,6S,7R,8R)-8-benzyl-3-{[(3-hydroxy-4-methoxypyridin-2-yl)carbonyl]amino}-6-methyl-4,9-dioxo-1,5-dioxonan-7-yl2-methylpropanoate(100 grams (g), 194 millimole (mmol)), DMAP (2.4 g, 19.4 mmol), andanhydrous acetonitrile (CH₃CN; 1000 mL). To this stirred suspension wasadded di-tert-butyl dicarbonate (BOC₂O; 93 g, 428 mmol) in portions.This mixture was stirred at ambient temperature for 1 hour (h), afterwhich N, N-diethylethylenediamine (24.8 g, 214 mmol) was added dropwiseover 20 minutes (min). The reaction was stirred at room temperature for2 days (d). The CH₃CN was removed on a rotary evaporator, and theresidue was dissolved in diethyl ether (Et₂O; 1500 mL). This solutionwas washed with saturated aqueous (aq) sodium bicarbonate solution(NaHCO₃; 500 mL), 0.1Normal (N) aq hydrogen chloride HCl (500 mL), water(500 mL) and saturated aq sodium chloride (NaCl) solution (brine; 500mL). The ether solution was dried over anhydrous magnesium sulfate(MgSO₄), filtered through a plug of silica gel, and concentrated undervacuum on a rotary evaporator. The resulting crude white solid (22 g)was dissolved in dichloromethane (CH₂Cl₂; 50 mL) and purified by silicagel flash chromatography (SiO₂, 0→100% ethyl acetate (EtOAc)/hexanes) toyield the title compound as a crystalline white solid (18 g, 20%): ¹HNMR (400 MHz, CDCl₃) δ 7.29-7.16 (m, 4H), 7.15-7.08 (m, 2H), 5.17 (t,J=9.7 Hz, 3H), 4.91 (dq, J=12.5, 6.3 Hz, 1H), 4.80 (s, 1H), 3.44 (s,1H), 3.02-2.84 (m, 2H), 2.62 (ddd, J=20.9, 18.0, 9.6 Hz, 2H), 1.60 (s,2H), 1.43 (s, 9H), 1.30 (d, J=6.3 Hz, 3H), 1.23 (dd, J=7.0, 2.2 Hz, 6H);¹³C NMR (101 MHz, CDCl₃) δ 175.62, 171.72, 137.93, 128.73, 128.57,126.66, 77.33, 77.02, 76.70, 75.05, 74.45, 51.94, 34.53, 34.11, 28.23,18.98, 18.96, 17.85; ESIMS m/z 462.5 ([M−H]⁻).

Example 1, Step 7b Preparation of(3S,6S,7R,8R)-8-benzyl-3-({[3-hydroxy-4-methoxy(2-¹⁴C)pyridin-2-yl]carbonyl}amino)-6-methyl-4,9-dioxo-1,5-dioxonan-7-yl2-methylpropanoate (Compound 8)

To a dry 50 mL round bottom flask equipped with magnetic stirrer, wascharged(3S,6S,7R,8R)-8-benzyl-3-[(tert-butoxycarbonyl)amino]-6-methyl-4,9-dioxo-1,5-dioxonan-7-yl2-methylpropanoate (570 milligrams (mg), 1.23 mmol) and 3.0 mL of 4Molar (M) HCl in dioxane. After 10 min, an additional 3.0 mL of dioxanewas added to facilitate stirring. The flask was sealed with a rubberseptum and stirred at room temperature for an additional 4 h. Theresulting thick, gelatinous mixture was concentrated by rotaryevaporation and the crude product was triturated in CH₂Cl₂ (10 mL) andagain concentrated in vacuo on a rotary evaporator. The residual whitesolid was suspended in CH₂Cl₂ (10 mL) and3-hydroxy-4-methoxy(2-¹⁴C)pyridine-2-carboxylic acid (274 mg, 1.59 mmol)was added in one portion. To this mixture was sequentially added4-methylmorpholine (746 mg, 7.38 mmol),2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (HATU; 701 mg, 1.84 mmol), and DMAP (15 mg, 0.123mmol), and the heterogeneous mixture was stirred at room temperatureunder a N₂ atmosphere for 16 h. The resulting clear, pale-yellowsolution was concentrated under a stream of N₂, and the residue wasdissolved in CH₂Cl₂ (5 mL) and purified by flash chromatography (SiO₂,100% EtOAc) to yield the title compound as a crystalline white solid(539 mg, 85%): structure conforms by HPLC (retention time match toauthentic unlabeled standard) and diode-array UV spectrum.

Example 1, Step 8 Preparation of(3S,6S,7R,8R)-8-benzyl-3-({[4-methoxy-3-{[(2-methylpropanoyl)oxy]methoxy}(2-¹⁴C)pyridin-2-yl]carbonyl}amino)-6-methyl-4,9-dioxo-1,5-dioxonan-7-yl2-methylpropanoate(Compound 9)

To a dry 50 mL round bottom flask equipped with magnetic stirrer and N₂inlet was charged(3S,6S,7R,8R)-8-benzyl-3-({[3-hydroxy-4-methoxy(2-¹⁴C)pyridin-2-yl]carbonyl}amino)-6-methyl-4,9-dioxo-1,5-dioxonan-7-yl2-methylpropanoate (479.5 mg, 0.928 mmol) and anhydrous acetone (20 mL).To this solution was added powdered sodium carbonate (Na₂CO₃; 98 mg,0.928 mmol) and the mixture was stirred at room temperature for 10 min,treated with iodomethyl isobutyrate (233 mg, 1.02 mmol) dropwise viasyringe, and the reaction mixture was stirred at ambient temperatureunder N₂ for 18 h. The acetone solution was filtered through a smallplug of SiO₂ and the filtrate was concentrated to dryness by rotaryevaporation. The crude yellow foam (765 mg) was dissolved in CH₂Cl₂ (2mL) and purified by flash chromatography (SiO₂, 70% EtOAc/hexanes) toyield the title compound as a white solid (455 mg, 79.5%): structureconforms by HPLC (retention time match to authentic unlabeled standard)and diode-array UV spectrum.

Example 2, Step 1 Preparation of(3S,6S,7R,8R)-8-benzyl-3-(tert-butoxycarbonylamino)-6-methyl-4,9-dioxo-1,5-dioxonan-7-ylisobutyrate (Compound 7)

To a suspension of(3S,6S,7R,8R)-8-benzyl-3-(3-hydroxy-4-methoxypicolinamido)-6-methyl-4,9-dioxo-1,5-dioxonan-7-ylisobutyrate (UK-2A; 10 g, 19.4 mmol) and DMAP (237 mg, 1.9 mmol) inCH₃CN (49 mL) was added BOC₂O (8.70 g, 39.8 mmol) and after a few minthe reaction became homogeneous. After 30 min,N,N-diethylethylenediamine was slowly added to the homogeneous solutionand stirring was continued at room temperature for 90 min. The CH₃CN wasremoved in vacuo and the resulting residue was dissolved in Et₂O (200mL) and washed with 1N HCl (100 mL). The phases were separated and theaqueous phase was extracted further with Et₂O (25 mL), and the combinedorganic phases were washed with 0.5N HCl, washed with saturated aqNaHCO₃, dried over MgSO₄, filtered, and concentrated to give a whiteglassy solid. Purification of the crude extract by flash chromatography(SiO₂; 0→3% EtOAc/CH₂Cl₂) gave the title compound as a white solid (2.41g, 27%): mp 143-145° C.; IR (neat) 1768, 1740, 1693, 1515 cm⁻¹; ¹H NMR(400 MHz, CDCl₃) δ 7.27-7.10 (m, 5H), 5.19 (m, 2H), 5.18 (dd, J=11.9,7.6 Hz, 1H), 4.91 (td, J=12.2, 6.0 Hz, 1H), 4.79 (s, 1H), 3.43 (s, 1H),3.00-2.86 (m, 2H), 2.68-2.58 (m, 2H), 1.43 (s, 9H), 1.30 (d, J=6.3 Hz,3H), 1.23 (d, J=7.0, Hz, 3H), 1.23 (d, J=7.0, Hz, 3H); ¹³C NMR (400 MHz,CDCl₃): 175.6, 171.7, 170.8, 154.7, 137.9, 128.7, 128.5, 126.6, 80.5,75.0, 74.4, 65.9, 51.9, 51.4, 34.5, 34.1, 28.2, 18.9, 17.8 ppm; ESIMSm/z 462.5 ([M−H]⁻).

Example 2, Step 2 Preparation of2-((3S,7R,8R,9S)-3-(tert-butoxycarbonylamino)-8-(isobutyryloxy)-9-methyl-2,6-dioxo-1,5-dioxonan-7-yl)aceticacid (Compound 10)

To a solution of(3S,6S,7R,8R)-8-benzyl-3-(tert-butoxycarbonylamino)-6-methyl-4,9-dioxo-1,5-dioxonan-7-ylisobutyrate (10 g, 21.57 mmol) in CH₃CN (72 mL), EtOAc (72 mL), andwater (575 mL) were added NaIO₄ (134 g, 626 mmol) and rutheniumtrichloride trihydrate (RuCl₃.3H₂O; 282 mg, 1.08 mmol). The reaction wasstirred at room temperature overnight. The resulting white suspensionwas diluted with water (700 mL) and extracted with CH₂Cl₂ (5×) and ethylacetate (2×). Activated carbon (12 g) was added to the combined organicextracts (around 2 L) and the mixture was stirred vigorously for 1 h.The mixture was filtered through Celite®, dried over MgSO₄, filtered,and concentrated to give the title compound as a gray solid (7.71 g,83%): mp 164-167° C.; IR (neat) 3375, 3293 (br), 1773, 1743, 1731, 1686,1157 cm⁻¹; ¹H NMR (300 MHz, CDCl₃) δ 5.30 (m, 2H), 5.04 (t, J=9.7 Hz,1H), 4.90 (m, 2H), 3.65 (s, 1H), 2.97 (m, 2H), 2.62 (m, 1H), 2.41 (m,1H), 1.45 (s, 9H), 1.29 (d, J=6.2 Hz, 3H), 1.22 (d, J=7.0 Hz, 6H); ¹³CNMR (101 MHz, CDCl₃) δ 175.78, 175.61, 171.53, 171.03, 154.88, 80.85,74.44, 74.05, 65.56, 51.16, 45.40, 34.03, 33.44, 28.24, 18.94, 18.83,17.77; ESIMS m/z 430.4 ([M−H]⁻).

Example 2, Step 3 Preparation of(3S,6S,7R,8R)-3-(tert-butoxycarbonylamino)-8-(iodomethyl)-6-methyl-4,9-dioxo-1,5-dioxonan-7-ylisobutyrate (Compound 11)

N-Methylmorpholine (2.5 mL, 23 mmol) and isobutylchloroformate (3.0 mL,23 mmol) were added sequentially to a 0° C. solution of2-((3S,7R,8R,9S)-3-(tert-butoxycarbonylamino)-8-(isobutyryloxy)-9-methyl-2,6-dioxo-1,5-dioxonan-7-yl)aceticacid (9.90 g, 23 mmol) in tetrahydrofuran (THF; 100 mL) in a roundbottom flask wrapped with aluminum foil. After 10 min, a solution of2-mercaptopyridine N-oxide (3.5 g, 27.5 mmol) and triethylamine (TEA;3.8 mL, 27.5 mmol) in THF (50 mL) was added slowly and the reactionstirred at 0° C. for 1.5 h. The precipitated N-methylmorpholinehydrochloride salt was removed by filtration through a Büchner funnelwith filter paper and the filtrate was concentrated in vacuo at roomtemperature. The oily residue was dissolved in CH₂Cl₂ (250 mL), iodoform(11.8 g, 30 mmol) was added, and the solution was placed in a UV reactorand irradiated with a 450 W Hg lamp with water cooling. After 1 h, theorange solution was removed from the reactor, treated with Celite®, andthe solvent was removed in vacuo. The adsorbed residue was partiallypurified by flash chromatography (SiO₂, 20% EtOAc/CH₂Cl₂) to give alight yellow solid. Further purification by flash chromatography (SiO₂,10% EtOAc/CH₂Cl₂) gave a dark orange solid, which was recrystallizedfrom EtOAc/hexane to yield the title compound as an off-white solid(5.99 g, 51%): mp 168-171° C.; IR (neat) 3386, 2977, 1771, 1760, 1742,1689, 1510 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ 5.36 (s, 1H), 5.21 (m, 1H),5.00 (t, J=9.7 Hz, 1H), 4.86 (m, 2H), 3.68 (brs, 1H), 3.31 (dd, J=11.5,9.3 Hz, 1H), 3.02 (m, 2H), 2.63 (m, 1H), 1.45 (s, 9H), 1.27 (d, J=6.3Hz, 3H), 1.22 (d, J=7.0 Hz, 6H); ESIMS m/z 536.3 ([M+Na]⁺).

Example 2, Step 4 Preparation of(3S,6S,7R,8R)-3-[(tert-butoxycarbonyl)amino]-6-methyl-4,9-dioxo-8-[(¹³C₆)phenylmethyl]-1,5-dioxonan-7-yl2-methylpropanoate(Compound 12)

A 7 mL vial equipped with magnetic stir bar and rubber septum (ovendried) was charged with zinc dust (471 mg, 7.2 mmol, ground with amortar and pestle prior to use) and anhydrous N,N-dimethylformamide(DMF; 0.70 mL). The mixture was treated with iodine (I₂, 183 mg, 0.72mmol) while stirring at room temperature under N₂ (exothermic). Afterstirring for 3 min, the vial was placed in an oil bath that had beenpre-heated to 40° C. and the reaction mixture was treated dropwise witha solution of(3S,6S,7R,8R)-3-((tert-butoxycarbonyl)amino)-8-(iodomethyl)-6-methyl-4,9-dioxo-1,5-dioxonan-7-ylisobutyrate (616 mg, 1.2 mmol) in warm DMF (1.20 mL). The reactionmixture was treated with a solution of (¹³C₆)iodobenzene ((¹³C₆)PhI; 300mg, 1.43 mmol,) in DMF (0.3 mL) followed bydichlorobis(tri-O-tolylphosphine)palladium (II) (PdCl₂[P(o-Tol)₃]₂; 94mg, 0.12 mmol). The vial was purged with N₂, the rubber septum wasreplaced with a Teflon® screw cap, and the reaction mixture was heatedat 40-50° C. for 30 min and then at 50-60° C. for 60 min. At this point,90 minutes post (¹³C₆)PhI addition, LC-MS showed only a minor amount ofthe reduced material and a major product with desired mass (around 71%).After stirring for an additional 60 min at 50-60° C., the reactionmixture was cooled to room temperature, filtered through a plug ofCelite®, and the plug was washed with EtOAc (around 30 mL). The filtratewas washed with H₂O (3×10 mL), washed with brine (1×10 mL), and theorganic phase was dried over sodium sulfate (Na₂SO₄), filtered, andconcentrated in vacuo to give a light yellow solid (0.59 g). The crudematerial was purified by flash chromatography (SiO₂, 0→50%EtOAc/hexanes) to give the title compound as a faint-yellow solid (0.377g, 68%): ¹H NMR (400 MHz, CDCl₃) δ 7.49-7.27 (m, 2H), 6.99 (m, 3H),5.28-5.11 (m, 3H), 4.97-4.72 (m, 2H), 3.43 (s, 1H), 3.02-2.83 (m, 2H),2.74-2.56 (m, 2H), 1.43 (s, 9H), 1.30 (d, J=6.3 Hz, 3H), 1.24 (d, J=2.1Hz, 3H), 1.22 (d, J=2.2 Hz, 3H); HRMS-ESI (m/z) [M+H]⁺ calcd for C₁₈¹³C₆H₃₃NO₈: 469.2407. found, 469.2428.

Example 2, Step 5 Preparation of(3S,6S,7R,8R)-3-{[(3-hydroxy-4-methoxypyridin-2-yl)carbonyl]amino}-6-methyl-4,9-dioxo-8-[(¹³C₆)phenylmethyl]-1,5-dioxonan-7-yl2-methylpropanoate(UK-2A-(¹³C₆)Ph, Compound 13)

A solution of(3S,6S,7R,8R)-8-(¹³C₆)phenylmethyl)-3-((tert-butoxycarbonyl)amino)-6-methyl-4,9-dioxo-1,5-dioxonan-7-ylisobutyrate (0.364 g, 0.775 mmol) in 1,4-dioxane (3 mL) was treated with4M HCl in 1,4-dioxane (3 mL, 12 mmol). The flask was “sealed” with a capand Parafilm® and stirred at room temperature for 20 h. The reactionmixture was concentrated in vacuo and the residue was triturated withCH₂Cl₂ and concentrated (5×10 mL). The resultant white solid was treatedwith CH₂Cl₂ (10 mL), 3-hydroxy-4-methoxypyridine-2-carboxylic acid(0.170 g, 1.008 mmol), HATU (0.442 g, 1.163 mmol), N-methylmorpholine(0.51 mL, 4.65 mmol) and a catalytic amount of DMAP. The slurrygradually turned into a clear, yellow solution. After 5 h, the reactionmixture was concentrated to one half the original volume under a streamof N₂, and the remaining solution was purified by flash chromatography(SiO₂, 0→100% EtOAc/hexanes) to give the title compound as a white solid(0.295 g, 74%): ¹H NMR (400 MHz, CDCl₃) δ 11.78 (s, 1H), 8.59 (d, J=8.1Hz, 1H), 7.99 (d, J=5.2 Hz, 1H), 7.58-7.27 (m, 2H), 7.17-6.91 (m, 3H),6.87 (d, J=5.2 Hz, 1H), 5.35 (s, 1H), 5.26-5.12 (m, 2H), 5.06-4.91 (m,1H), 3.94 (s, 3H), 3.62 (s, 1H), 3.06-2.88 (m, 2H), 2.78-2.68 (m, 1H),2.62 (dt, J=14.0, 7.0 Hz, 1H), 1.33 (d, J=6.3 Hz, 3H), 1.25 (d, J=2.3Hz, 3H), 1.23 (d, J=2.3 Hz, 3H); HRMS-ESI (m/z) [M+H]⁺ calcd for C₂₀¹³C₆H₃₀N₂O₉: 520.2153. found, 520.2167.

Example 2, Step 6 Preparation of{[4-methoxy-2-({(3S,7R,8R,9S)-9-methyl-8-[(2-methylpropanoyl)oxy]-2,6-dioxo-7-[(¹³C₆)phenylmethyl]-1,5-dioxonan-3-yl}carbamoyl)pyridin-3-yl]oxy}methyl2-methylpropanoate (Compound 14)

A solution of(3S,6S,7R,8R)-3-{[(3-hydroxy-4-methoxypyridin-2-yl)carbonyl]amino}-6-methyl-4,9-dioxo-8-[(¹³C₆)phenylmethyl]-1,5-dioxonan-7-yl2-methylpropanoate(273 mg, 0.531 mmol) in anhydrous acetone (4 mL) was treated withpowdered potassium carbonate (325 mesh K₂CO₃, 147 mg, 1.06 mmol). Theslurry was treated with a solution of iodomethyl isobutyrate (145 mg,0.637 mmol) in acetone (2 mL) while stirring at room temperature underN₂. After stirring for 5 h, the reaction mixture was filtered through aplug of Celite® and the plug was washed with EtOAc (30 mL). The filtratewas washed with H₂O (2×10 mL), washed with brine (1×10 mL), and theorganic phase was dried over Na₂SO₄, filtered and concentrated in vacuoto give 0.445 g of a light yellow oil. The oil was purified by flashchromatography (SiO₂, 0→100% EtOAc/hexanes) to give the title compoundas an off-white solid (0.229 g, 70%): ¹H NMR (400 MHz, CDCl₃) δ 8.53 (d,J=7.9 Hz, 1H), 8.26 (d, J=5.4 Hz, 1H), 7.50-7.28 (m, 2H), 7.13-6.97 (m,3H), 6.95 (d, J=5.4 Hz, 1H), 5.79-5.71 (m, 2H), 5.36 (s, 1H), 5.26-5.13(m, 2H), 5.05-4.92 (m, 1H), 3.89 (s, 3H), 3.59 (s, 1H), 3.06-2.88 (m,2H), 2.71 (s, 1H), 2.66-2.58 (m, 1H), 2.53 (dt, J=14.0, 7.0 Hz, 1H),1.31 (d, J=6.3 Hz, 3H), 1.25 (d, J=2.2 Hz, 3H), 1.23 (d, J=2.2 Hz, 3H),1.13 (d, J=7.0 Hz, 6H); HRMS-ESI (m/z) [M+H]⁺ calcd for C₂₅¹³C₆H₃₈N₂O₁₁: 620.2677. found, 620.2681.

Example 3, Step 1 Preparation of(3S,6S,7R,8R)-3-[(tert-butoxycarbonyl)amino]-6-methyl-4,9-dioxo-8-[phenyl(UL-¹⁴C)methyl]-1,5-dioxonan-7-yl2-methylpropanoate (Compound 15)

A 7 mL vial equipped with magnetic stir bar and rubber septum (ovendried) was charged with zinc dust (605 mg, 9.25 mmol, ground with amortar and pestle prior to use) and anhydrous DMF (0.80 mL). The mixturewas treated with I₂ (235 mg, 0.92 mmol) while stirring at roomtemperature under N₂ (exothermic). After stirring for 3 min, the vialwas placed in an oil bath that had been pre-heated to 40° C. and thereaction mixture was treated dropwise with a solution of(3S,6S,7R,8R)-3-((tert-butoxycarbonyl)amino)-8-(iodomethyl)-6-methyl-4,9-dioxo-1,5-dioxonan-7-ylisobutyrate (791 mg, 1.54 mmol) in warm DMF (1.50 mL). After stirring at40° C. for 20 min, the reaction mixture was treated with a solution of(UL-¹⁴C)iodobenzene (50.000 millicuries (mCi), 1.85 mmol) in DMF (0.4mL) followed by PdCl₂[P(o-Tol)₃]₂ (121 mg, 0.154 mmol). The vial waspurged with N₂, the rubber septum was replaced with a Teflon® screw cap,and the reaction mixture was heated at 40-50° C. for 30 min and then at50-60° C. for 120 min. The reaction mixture was cooled to roomtemperature and filtered through a plug of Celite®, and the plug waswashed with EtOAc (30 mL). The filtrate was washed with H₂O (3×10 mL),washed with brine (1×10 mL), and the organic phase was dried by passingthrough a plug of Na₂SO₄/MgSO₄ in a 10 mL disposable pipet. The filtratewas concentrated in vacuo to give a light-yellow solid (0.778 g). Thesolid was purified by flash chromatography (SiO₂, 0→50% EtOAc/hexanes)to give the desired product as a peach colored solid (0.620 g, 87%):FTMS(ESI⁺) 464 (M+H), 416, 408, 364. HPLC analysis of the solid showed95% chemical (UV at 254 nm) and approximately 90% radiochemical purity(β-RAM). The retention time matched an unlabeled standard of thismaterial.

Example 3, Step 2 Preparation of(3S,6S,7R,8R)-3-{[(3-hydroxy-4-methoxypyridin-2-yl)carbonyl]amino}-6-methyl-4,9-dioxo-8-[phenyl(UL-¹⁴C)methyl]-1,5-dioxonan-7-yl2-methylpropanoate(UK-2A-(UL-¹⁴C)Ph, Compound 16)

The(3S,6S,7R,8R)-8-((UL-¹⁴C)phenylmethyl)-3-((tert-butoxycarbonyl)amino)-6-methyl-4,9-dioxo-1,5-dioxonan-7-ylisobutyrate (0.620 g, 1.34 mmol) was dissolved in 1,4-dioxane (5 mL) andtransferred to a 50 mL round bottom flask equipped with a magnetic stirbar. The resultant solution was treated with 4M HCl in 1,4-dioxane (5mL, 20 mmol) while stirring at room temperature under N₂. After stirringfor 20 h at room temperature, the reaction mixture was concentrated invacuo. The residue was triturated with CH₂Cl₂ and concentrated (5×10mL). The resultant white solid was suspended in CH₂Cl₂ (10 mL), andtreated with 4-methoxy-3-hydroxypicolinic acid (0.294 g, 1.74 mmol),HATU (0.763 g, 2.01 mmol, 1.5 equiv,), N-methylmorpholine (0.88 mL, 8.03mmol, 15 equiv.) and a catalytic amount of DMAP. The slurry graduallyturned into a clear, yellow solution. After stirring for 22 h at roomtemperature, the reaction mixture was concentrated to about one half theoriginal volume under a stream of N₂. The remaining solution waspartially purified by flash chromatography (SiO₂, 0→100% EtOAc/hexanes)to give 0.453 g of a white solid. HPLC analysis indicates that this iscontaminated with 15% of an non-radioactive impurity. This solid wasagain subjected to the flash chromatography conditions to give the titlecompound as a white solid (0.336 g, 49%): FTMS(ESI⁺) 515 (M+H). HPLCanalysis of the solid showed >98% chemical (UV at 254 nm) andradiochemical purity (β-RAM). The HPLC retention time and UV spectrummatched an unlabeled standard of UK-2A.

Example 3, Step 3 Preparation of{[4-methoxy-2-({(3S,7R,8R,9S)-9-methyl-8-[(2-methylpropanoyl)oxy]-2,6-dioxo-7-[phenyl(UL-¹⁴C)methyl]-1,5-dioxonan-3-yl}carbamoyl)pyridin-3-yl]oxy}methyl2-methylpropanoate (Compound 17)

A solution of UK-2A-(UL-¹⁴C)Ph (294 mg, 0.571 mmol) in anhydrous acetone(8 mL) was treated with powdered K₂CO₃ (111 mg, 0.800 mmol, 325 mesh).The resultant mixture was treated with a solution of iodomethylisobutyrate (156 mg, 0.686 mmol) in acetone (2 mL) while stirring atroom temperature under N₂. After stirring for 17 h, the reaction mixturewas treated with an additional 33 microliters (μL) of iodomethylisobutyrate (0.143 mmol, 25 mol %). After an additional 4 h, HPLCanalysis showed little change. The reaction mixture was filtered througha plug of Celite®/silica gel, washing with EtOAc, and the filtrate wasconcentrated in vacuo to give a light-yellow foam (0.497 g). The crudematerial was purified by flash chromatography (SiO₂, 0→25% EtOAc/CH₂Cl₂)and the purified material was treated with Et₂O and concentrated invacuo (3×3 mL). The resultant solid was dried to constant weight underhigh vacuum (40-50° C.) to give the desired product as a white solid(215 mg, 61%): FTMS(ESI⁺) 615 (M+H). The total radioactivity was foundto be 10.182 mCi with a specific activity of 29.1 mCi/mmol. HPLCanalysis of the solid showed >98% chemical (UV at 254 nm) andradiochemical purity (β-RAM). The HPLC retention time and UV spectrummatched an unlabeled standard of Compound 17.

While the novel technology has been illustrated and described in detailin the figures and foregoing description, the same is to be consideredas illustrative and not restrictive in character, it being understoodthat only the preferred embodiments have been shown and described andthat all changes and modifications that come within the spirit of thenovel technology are desired to be protected. As well, while the noveltechnology was illustrated using specific examples, theoreticalarguments, accounts, and illustrations, these illustrations and theaccompanying discussion should by no means be interpreted as limitingthe technology. All patents, patent applications, and references totexts, scientific treatises, publications, and the like referenced inthis application are incorporated herein by reference in their entirety.

What is claimed is:
 1. A radiolabeled compound of the following formula:

Wherein R₁ is selected from the group consisting of: H and

R₂ is selected from the group consisting of OH and

and R₄ is selected from the group consisting of Ph, (¹³C₆)Ph, and(UL-¹⁴C)Ph.
 2. The compound according to claim 1, wherein R₁ is


3. The compound according to claim 1, wherein R₂ is:


4. The compound according to claim 1, wherein R₁ is H.
 5. The compoundaccording to claim 1, wherein the compound is:


6. An isotopically labeled compound of the following formula:

wherein R₃ is selected from the group consisting of: H,

and R₄ is selected from the group consisting of (¹³C₆)Ph and (UL-¹⁴C)Ph.7. The compound according to claim 6, wherein R₃ is:


8. The compound according to claim 6, wherein R₃ is:


9. The compound according to claim 6, wherein R₃ is:


10. The compound according to claim 6, wherein R₄ is (¹³C₆)Ph.
 11. Thecompound according to claim 6, wherein R₄ is (UL-¹⁴C)Ph.
 12. A methodfor studying the biosphere, comprising the steps of: exposing samples toan isotopically labeled compound selected from the group consisting of(a) and (b), wherein:

 wherein R₁ is selected from the group consisting of H and

 R₂ is selected from the group consisting of OH and

 and R₄ is selected from the group consisting of Ph, (¹³C₆)Ph, and(UL-¹⁴C)Ph; and

 wherein R₃ is selected from the group consisting of:

 and R₄ is at least one atom selected from the group consisting ofPh-¹³C₆ and Ph-UL-¹⁴C; and determining which samples include anisotopically labeled signature from said compound.
 13. The methodaccording to claim 12, wherein the compound is (a) and the sample is aportion of a plant.
 14. The method according to claim 12, wherein thecompound is (a) and the sample is from a surface adjacent to a plant.15. The method according to claim 12, wherein the compound is (a) andthe sample is from a material that is in communication with a surfaceadjacent to a plant.
 16. The method according to claim 12, where thecompound is (a).
 17. The method according to claim 12, wherein thecompound is (b) and the sample is a portion of a plant.
 18. The methodaccording to claim 12, wherein the compound is (b) and the sample isfrom a surface adjacent to a plant.
 19. The method according to claim12, wherein the compound is (b) and the sample is from a material thatis in communication with a surface adjacent to a plant.
 20. The methodaccording to claim 12, where the compound is (b).